We propose to develop a deeper understanding of the structural basis for the behavior/function of the opioid receptor subfamily of G protein-coupled receptors (GPCR). Structures of all three opioid receptors: ? -opioid receptor (MOR), ? -opioid receptor (KOR), ?-opioid receptor (DOR), and the opioid-like nociceptin/orphanin FQ opioid-like receptor (NOP), have recently been solved. This breakthrough created a unique opportunity to pursue a comprehensive understanding of ligand recognition and selectivity, as well as structural mechanisms of opioid receptor activation and biased signaling. Attaining this goal will require solving a large number of structures of ligand-receptor complexes similar to what is routinely done in structure based drug design targeting soluble proteins. This is now possible since our GPCR Structure Determination Pipeline (GSDP) and our newly developed GPCR Protein Fusion Toolchest has enabled large-scale structure determination of ligand-receptor complexes. The GSDP has been used by us in the successful structure determination of 14 different human GPCRs and their complexes, including two new receptors in activated states with bound agonist. In this proposed study, we will conduct similar efforts toward the crystallization and structural determination of ligand-receptor complexes of all of the receptors in the opioid family using agonists, antagonists, inverse agonists, and allosteric modulators. Criteria for opioid receptor and signaling pathway selectivity will be approached by attempting structure determination of several complexes of functional near-wild type constructs and rationally designed mutants. Within three specific aims we will solve several ligand-receptor structures on (1) KOR, (2) NOP, and (3) MOR and DOR.